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Pathology and Immunohistochemistryof Hereditary Colorectal CancerMelissa W. Taggart, MD, and Asif Rashid, MD, PhD

Hereditary colorectal adenocarcinomas only represent a small proportion of all colorectalcancers and, of those, only a fraction have distinct clinical or pathologic features and amutation of a gene associated with a hereditary syndrome. These hereditary conditionsinclude hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis,MUTYH-associated polyposis, Peutz–Jeghers syndrome, and juvenile polyposis syndrome.Histopathologic features in patients with colorectal lesions (polyps, adenocarcinoma) thatsuggest the presence of a hereditary condition include the number and type of polyps, themorphologic features of the colorectal carcinoma, and the presence of extracolonic lesionsor tumors. In addition, immunohistochemical stains can assist in identifying patients withhereditary nonpolyposis colorectal cancer.

Semin Colon Rectal Surg 22:71-76 © 2011 Elsevier Inc. All rights reserved.

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It is estimated that approximately 20% of colorectal carci-nomas are heritable. Nevertheless, only a small fraction

(around 5%) of these patients has distinct clinical and/orpathologic features which suggest a familial syndrome asso-ciated with an increased incidence of colorectal adenocarci-noma.1-3 Generally, a heritable cancer is suspected whenthere is a family history of colorectal or other cancers,younger age of onset, increased number or uncommon his-tologic type of polyps, certain extracolonic lesions, or syn-chronous or metachronous colorectal or extracolonic tu-mors. Confirmation is made with adjunctive moleculartesting.4,5 Unfortunately, for the majority of patients withamilial clustering of colon cancer, the genetic defect has notet been defined. These are high-risk colon cancers fromndetermined gene(s) and hence not associated with thenown syndromes. These are labeled familial colorectal can-er type X or syndrome X and are the result of low-pen-trance loci or genetic polymorphisms.6

Syndromic familial colorectal adenocarcinoma can bebroadly classified by the presence or absence of intestinalpolyps and the type of polyp (adenomatous or hamartoma-tous) (Table 1). Syndromes that are associated with an in-creased number of adenomatous polyps include classic famil-ial adenomatous polyposis (cFAP), attenuated familial

Department of Pathology, The University of Texas, M.D. Anderson CancerCenter, Houston, TX.

Address reprint requests to: Melissa W. Taggart, MD, Department of Pathol-ogy, The University of Texas, M.D. Anderson, Cancer Center, 1515Holcombe Blvd., Unit 085, Houston, TX 77030. E-mail: mtaggart@

mdanderson.org

1043-1489/$-see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1053/j.scrs.2010.12.003

adenomatous polyposis (aFAP), and MUTYH-associated pol-yposis (MAP). Increased colorectal adenocarcinoma is alsoseen in some of the hamartomatous polyposis syndromes—Peutz–Jeghers syndrome (PJS) and juvenile polyposis syn-drome (JPS). Paradoxically, hereditary nonpolyposis colorec-tal cancer (HNPCC, Lynch syndrome) can have adeno-matous polyps and may cause confusion with aFAP andMAP. These syndromes may also have characteristic extraco-lonic lesions that distinguish or subcharacterize the syn-dromes. In some HNPCC patients, the histopathologic char-acteristics of the colorectal cancer may suggest the syndrome.

HereditaryNonpolyposis ColorectalCancer (Lynch Syndrome)HNPCC (Lynch syndrome) accounts for 2% to 5% of all colo-rectal carcinoma.7-9 HNPCC is an autosomal-dominant con-

ition characterized by a germ line mutation in one of theNA mismatch repair (MMR) genes, MLH1, MSH2, MSH6, or

PMS2. The function of DNA mismatch enzymes is to repairsingle nucleotide base mismatches (errors) that occur duringDNA replication which are especially prevalent in the repeatnucleotide sequences (microsatellites). Loss of DNA mis-match enzyme function results in new alleles for the locus(microsatellite instability). Microsatellite instability in colo-rectal cancer or other lesions can be classified as low or high,depending on the amount of microsatellite instability (lessthan or more than 40% of markers, respectively).10 Most

colorectal adenocarcinomas in HNPCC show high levels of

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72 M.W. Taggart and A. Rashid

microsatellite instability. Around 15% of sporadic cancersalso are microsatellite instability-high, predominately be-cause of methylation of MLH1 promoter.

Colorectal adenocarcinomas in HNPCC patients are morecommon in the right colon and have a younger age of onset.HNPCC-associated and sporadic microsatellite instability-highcolorectal adenocarcinomas can be histologically indistinguish-

Table 1 Features of Hereditary Colorectal Carcinoma Syndro

Syndrome Gene(s) Polyp Type

NPCC*urcot†

Muir-Torre†

MLH1MSH2MSH6PMS2

Adenomatous

FAP*Turcot†

Gardner†

APC Adenomatous cFAP >100polyps, aFAP <100 polyps

MAP* MUTYH Adenomatous, serrated

JS* STK11 Hamartomatous,Peutz–Jegher type

JPS*HHT†

SMAD4BMPR1A

Hamartomatous, juvenile typ

*CRC, colorectal carcinoma; HNPCC, hereditary nonpolyposis colopolyposis; cFAP, classic familial adenomatous polyposis; aFAP, aof the retinal pigment epithelium; MAP, MUTYH-associated polyptubules; JPS, juvenile polyposis syndrome.

Turcot syndrome: colorectal cancer and brain tumor (HNPCC—glsyndrome: defect in mismatch repair gene (primarily MSH2) witmalignancy (usually gastrointestinal or genitourinary); Gardner syosteomas, and cutaneous lesions; HHT (hereditary hemorrhagicder associated with SMAD4 mutations.

able from sporadic microsatellite-stable adenocarcinoma (well-

formed glands composed of cells with pseudostratified nucleiand luminal necrosis). Nevertheless, many (approximately50%) microsatellite instability-high tumors show distincthistologic features that are characteristic but not specific.These include signet ring cell, mucinous, poorly differenti-ated, or solid sheet (medullary) types of morphology; fur-thermore, tumors with variegated morphology (combination

CRCCharacterization

ExtracolonicLesions/Tumors

istologic types: signetring cell mucinouspoorly differentiated/solid variegatedtumor-associatedlymphocytes: tumorinfiltratinglymphocytesCrohn-like reaction

Carcinoma: endometrium (mostfrequent), stomach, ovary,biliary tree, upper urinarytract (urothelial carcinomas),pancreas, small intestine

CNS* tumors (glioblastomamultiforme)

onventional CHRPE*Skin and soft tissue lesionsOsteomaDesmoid tumorFundic gland polypSmall intestine/ampullary/

gastric adenomasCarcinoma:

Duodenum/periampullary,stomach, pancreas, thyroid

HepatoblastomaCNS tumors (medulloblastoma,

astrocytoma)onventional Fundic gland polyp

Duodenal adenomaBenign skin, endometrial and

breast tumorsCarcinoma: Duodenal,

gynecologic tract, bladder,skin, breast

onventional Skin pigmentationOvarian SCTAT*Testicular large-cell calcifying

Sertoli cell tumorsCarcinomas: Small intestine,

stomach, breast, pancreas,ovary, cervix

onventional Gastric and small intestinalhamartomatous polyps

Carcinoma: stomach, smallintestine, pancreas

cancer; CNS, central nervous system; FAP, familial adenomatoused familial adenomatous polyposis; CHRPE, congenital hypertrophyJS, Peutz–Jeghers syndrome; SCTAT, sex cord tumor with annular

oma multiforme; FAP—medulloblastoma, astrocytoma); Muir-Torreceous neoplasms, with or without keratoacanthomas, and viscerale: FAP with extragastrointestinal lesions, including desmoid tumors,ectasia/Osler-Weber-Rendu disease): vascular malformation disor-

mes

H

C

C

C

e C

rectalttenuatosis; P

ioblasth sebandromtelangi

of different morphologic types) and tumors with an associ-

q

Pathology and immunohistochemistry of hereditary colorectal cancer 73

ated increase in lymphocytes (either intratumoral lympho-cytes and/or Crohn-like reaction) are characteristic.8,11,12 Sig-net ring cell adenocarcinomas are distinguished bycharacteristic dyshesive single cells with a large mucin drop-let that indents the nucleus that comprise most of the tumor.Mucinous (colloid) adenocarcinomas are adenocarcinomasin which most of the tumor is composed of large pools ofmucin, which either is lined by or contains neoplastic cellswith variable epithelial dysplasia. Another characteristicmorphology of microsatellite instability-high tumors is asolid sheet of neoplastic cells that are arranged in a syncytialpattern (ie, without distinct cell borders) and lack significantgland formation. Occasionally, microsatellite instability-hightumors show a mixture of these different morphologic pat-terns (variegated morphology), including conventionalgland-forming adenocarcinoma. Lymphocytes may be prom-inent, either with numerous lymphocytes intimately associ-ated with and infiltrating the neoplastic cells (tumor infiltrat-ing lymphocytes) (Fig. 1A) or as aggregates of lymphocytes innonneoplastic tissue that confront the tumor at its advancingborder (Crohn-like reaction) (Fig. 1B).

Immunohistochemistry is used to identify specific proteincell markers using targeted antibodies tagged with a chromo-gen and can be applied to sections of paraffin-embedded

Figure 1 (A) Lymphocytes are not only present in the str(tumor-infiltrating lymphocytes) (Hematoxylin and eosrowheads) are present near the leading of the tumor (Cro�20). (C) Immunohistochemical stain for MLH1 showintervening stromal cells have preserved expression o�200). (D) In the same adenocarcinoma, an immunohisin both the neoplastic glands and the surrounding stro�200).

tissue. Immunohistochemical stains for the DNA MMRs can a

be employed as screening tests for HNPCC. Loss of immuno-staining corresponds to a loss of protein expression and sug-gests a microsatellite instability-high phenotype. Although itdoes not distinguish a germ line mutation (as seen in HN-PCC) from a sporadic adenocarcinoma, its sensitivity is 92%for detecting microsatellite instablity-high tumors when all 4antibodies (MLH1, MLH2, MSH6, and PMS2) are used (Fig.1C and 1D).13 The MMR proteins form two heterodimersconsisting of one of the two major enzymes (MLH1 or MLH2),which pairs with a respective minor enzyme (PMS2 or MSH6,others). The loss of enzyme function can occur in any of thefour components; however, loss of one of the major proteinswill result in a loss of its respective minor protein but not viceversa. For example, the loss of staining with MLH1 will alsoresult in a loss of staining with PMS2 but the reverse is nottrue (PMS2 can be lost without loss of MLH1).

HNPCC is most frequently due to mutations of MLH1 andMSH2 and infrequently to mutations of MSH6 and PMS2.9

Sporadic microsatellite instability-high colorectal cancers aregenerally the result of methylation of MLH1, which alsoshows a loss of expression of MLH1 and PMS2. Thus, furtherancillary molecular studies are added, such as BRAF muta-tion analysis or MLH1 methylation. These changes are fre-uent in sporadic tumors but infrequent in HNPCC tumors,

ound the neoplastic glands but also infiltrate the glands, magnification, �400). (B) Lymphoid aggregates (ar-reaction) (Hematoxylin and eosin stain, magnification,ss of nuclear expression in an adenocarcinoma. The1 (MLH1 immunohistochemical stain, magnification,ical stain for MSH2 shows preserved nuclear expressionls (MSH-2 immunohistochemical stain, magnification,

oma arin stainhn-likeing lo

f MLHtochemmal cel

nd—in conjunction with family history—can help to dis-

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74 M.W. Taggart and A. Rashid

tinguish sporadic and HNPCC tumors. Immunohistochemi-cal stains also assist in identifying the affected gene in HN-PCC patients, thus, theoretically, limiting confirmatorymutational analysis to one gene.

Frequently, when the histologic features of a carcinoma inthe colorectum or in the metastasis are not classic, immuno-histochemical stains are used to clarify whether the tumor isof primary or secondary nature (metastasis). Immunohisto-chemical stains commonly used for this purpose include cy-tokeratin 7, cytokeratin 20, and CDX2. Most colorectal car-cinomas are positive for cytokeratin 20, negative or onlyfocally positive for cytokeratin 7, and diffusely and stronglypositive for CDX2. Occasionally, microsatellite instability-high colorectal adenocarcinomas are positive for cytokeratin7 and negative for cytokeratin 20 and CDX2.14 Awareness ofhis phenomenon may direct the pathologist to perform fur-her immunohistochemical stains for DNA MMR proteins.

Patients with HNPCC also have an increased risk of ade-ocarcinomas in other sites, including endometrium (mostrequent), stomach, ovary, biliary tree, upper urinary tracturothelial carcinomas), pancreas, and small intestine.4,15 Inome sites (eg, pancreas), tumors can have similar features ashe colonic adenocarcinomas.16 Sebaceous neoplasms, with

or without keratoacanthomas, in conjunction with a visceral

Figure 2 (A) Colectomy specimen from a patient withcharacterized as adenomatous. (B) Three sessile tubularwith FAP. The cells composing the adenomas have hypmagnification, �20). (C) Monocryptal adenoma (dysplahyperchromatic, crowded, and enlarged nuclei, is presestain, magnification, �100). (D) Desmoid tumor compoeosin stain, magnification, �100).

malignancy (usually gastrointestinal or genitourinary) is p

found in Muir–Torre syndrome, which is commonly associ-ated with a mutation in a MMR gene (primarily MSH2).

FamilialAdenomatous Polyposis (FAP)FAP is caused by an autosomal-dominant mutation in theadenomatous polyposis coli gene.17,18 Patients with classicFAP generally have more than 100 grossly detectable colonicadenomatous polyps (Fig. 2A). aFAP has less than 100 co-lonic polyps, which are often in a more proximal loca-tion.19,20 Microscopically, the colonic polyps in cFAP or aFAPare adenomas (epithelial proliferation showing dysplasia)(Fig. 2B) and can vary in size from monocryptal adenomas(dysplastic epithelium which involving a single crypt) (Fig.2C) to larger polyps indistinguishable from sporadic adeno-mas. Characteristically, in cFAP, and less commonly aFAP,patients have polyps in other gastrointestinal sites, includinggastric fundic gland polyps and gastric and small intestinaladenomatous polyps, especially duodenal and ampullary ad-enomas.21 Fundic gland polyps are proliferations of the gas-ric fundic glands, which are composed of chief and parietalells and some foveolar-type cells, with associated gland dil-tation. Although these polyps are common in the general

owing numerous polyps, which microscopically wereas (arrowheads) in one section of colon from a patient

matic elongated nuclei (Hematoxylin and eosin stain,thelium which involving single crypt), characterized byhe colon of a patient with FAP (Hematoxylin and eosinerlacing fascicles of bland fibroblasts (Hematoxylin and

FAP shadenomerchro

stic epint in tsed int

opulation, especially in patients on proton-pump inhibitor

pchc

w

i

Pathology and immunohistochemistry of hereditary colorectal cancer 75

therapy, patients with FAP usually have multiple fundicgland polyps and roughly half of these polyps have foveolardysplasia. Other extraintestinal lesions and malignancies canbe seen in FAP patients (Table 1).3 The term Gardner syn-drome applies to FAP patients with extragastrointestinal le-sions, including desmoid tumors (Fig. 2D), osteomas, usu-ally found in the skull bones and mandible, and cutaneouslesions (fibromas, lipomas, and sebaceous and epidermoidcysts).22,23 Colorectal cancer and brain tumor is termed Tur-cot syndrome and interestingly can be associated with muta-tions of the APC gene (medulloblastoma, astrocytoma) ormutations of mismatch repair genes (glioblastoma multi-forme).

MUTYH-Associated PolyposisMUTYH-associated polyposis (also known as MYH-associ-ated polyposis or MAP) is an autosomal-recessive conditiondue to biallelic mutations in the MUTYH gene.24 The colonic

olyp load mimics attenuated FAP. Although most of theolonic polyps are adenomatous, serrated polyps (includingyperplastic polyps and sessile serrated polyps/adenomas)an also be increased.25 A minority of these patients can also

have an increased incidence of duodenal and gastric polypsas well as of other benign and malignant tumors in duode-num, gynecologic organs (endometrial and ovarian), blad-der, skin, and breast.26,27

Peutz–Jeghers SyndromePJS is distinguished by hamartomatous polyps of the Peutz–Jeghers type and is caused by a mutation in serine–threoninekinase 11 (STK11) gene.28 These polyps generally occur inthe small intestine and the colon, and in some patients, canalso develop in the stomach. The characteristic feature of thePeutz–Jeghers polyps is the arborizing smooth muscle layerthat extends into the lamina propria and forms a villiformpolyp (Fig. 3A). The epithelium is usually nondysplastic butcan show varying degrees of dysplasia. Larger polyps canresult in displacement of mucosal elements into the deeperlayers of the intestine, which can mimic invasive adenocarci-noma.

The most common extracolonic malignancies in PJS pa-tients include small intestinal, gastric, breast, gynecologic,and pancreas cancers.29,30 Benign ovarian sex cord tumors

ith annular tubules can be seen in females with PJS.31 Malescan develop calcifying testicular large-cell Sertoli cell tu-mors.32

Juvenile Polyposis SyndromeJPS is caused by a mutation in SMAD4 or BMPR1A.33 As thename indicates, the characteristic polyp seen in JPS is thejuvenile polyp; however, these polyps are not necessarilyspecific and can be found in non-syndromic patients. Classi-cally, juvenile polyps are spherical polypoid lesions with cys-tic glands embedded in an edematous and inflamed stroma

(Fig. 3B). The surface may be eroded, and these polyps can

auto-amputate and be excreted with the feces. Occasionally,juvenile polyps show a more arborizing architecture withincreased smooth muscle extension into the lamina propria,hence mimicking Peutz–Jeghers polyps. Varying degrees ofdysplasia can be seen in these polyps. Juvenile polyps aremost frequently located in the colon but can also occur in thesmall intestine and stomach. Because of overlapping features,juvenile polyps can resemble inflammatory polyps in boththe colon and the stomach. Patients with SMAD4 mutationsmay also have hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease).34 There is an increased risk for smallntestinal and gastric cancers in JPS patients.33

ConclusionsPathologic features that suggest a hereditary syndrome in-clude exaggerated number of sporadically occurring lesions,such as adenomatous polyps, unusual (hamartomatous) pol-yps, distinctive histologic features of the colorectal carcino-

Figure 3 (A) Peutz–Jeghers hamartomatous polyp of the small intes-tine with characteristic arborizing smooth muscle (Hematoxylinand eosin stain, magnification, �20). (B) Juvenile hamartomatouspolyp with eroded surface, dilated colonic glands, and inflamedstroma (Hematoxylin and eosin stain, magnification, �40).

mas, and some immunohistochemical findings. Extracolonic

2

2

2

2

2

2

2

2

2

2

3

3

3

3

3

76 M.W. Taggart and A. Rashid

manifestations also give clues to syndromes that have an in-creased incidence of colorectal adenocarcinoma.

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